preliminary evidence of gene-exposure interaction in 1991 gulf … · preliminary evidence of...

Butyrylcholinesterase in Relation to Gulf War Illness:

Preliminary Evidence of Gene-Exposure Interaction in 1991 Gulf War Veterans

Lea Steele, Ph.D.

Director, Veterans Health Research Baylor Institute of Biomedical Studies

RAC-GWVI April 14, 2015

51

1990-1991 Gulf War: Operations Desert Shield/Desert Storm

Aug 2, 1990 - Iraq invaded Kuwait

Jan 16, 1991 - Air strikes began

Feb 24, 1991 - Ground combat began

Feb 28, 1991 - Cease fire declared

________________

- 6 weeks air strikes, 4 day ground war

- 37 countries in Allied Coalition

~ 700,000 U.S. troops

- Decisive victory; relatively few casualties

Appendix A Presentation 1 - Lea Steele

RAC-GWVI Meeting Minutes April 20-21, 2015

Page 50 of 279

52

1991: Returning Veterans Report Difficult Symptoms, Not Explained by Familiar Medical or Psych Diagnoses

“Gulf War Syndrome:” Widespread reports of persistent health problems

- Chronic headaches

- Widespread pain

- Memory and concentration difficulties

- Persistent, unexplained fatigue

- Chronic diarrhea

- Respiratory problems

- Mood changes

- Unusual skin rashes

What Caused Gulf War Illness? Early on, many suspected causes/contributors



Page 51 of 279

Oil Fires: Over 600 burning wells Feb-Nov 1991

Chemical Weapons



Page 52 of 279

DOD models indicate 100,000 Gulf war troops potentially exposed to nerve agents resulting from Khamisiyah demolitions

PB: Pyridostigmine Bromide

(anti-nerve gas pills)



Page 53 of 279

INFECTIOUS DISEASES

Widespread use, overuse of pesticides and insect repellants



4 of 279

60

Large number of Gulf War-related experiences/exposures of potential concern

— Psychological stress, trauma

— Chemical weapons

— Oil well fires

— Munitions containing depleted uranium

— Heavy use of insecticides/repellants

— PB pills (pyridostigmine bromide)

— Vaccines

— Infectious diseases

— Tent heaters

— Particulates

— Fuel exposures

— Solvents, CARC paint

61

Association of Deployment Experiences/Exposures with Gulf War Illness Synthesis of Evidence from Epidemiologic Studies of Gulf War Veterans

- Psychological stressors

Evidence indicates no association

- Pyridostigmine bromide (PB) - Pesticides

Evidence consistently indicates a significant association

- Low-level nerve agents - Sustained oil well smoke - Large number of vaccines - Combinations of exposures

Unclear, association cannot be ruled out; evidence is inconsistent or limited in important ways

- Depleted uranium - Anthrax vaccine - Fuels, solvents - Sand, particulates - Other

Little evidence of association; unlikely to have been primary contributing factor for the majority of affected veterans

Adapted from : Research Adv. Cmte Gulf War Illnesses: Gulf War Illness and the Health of Gulf War Veterans, 2008



Page 55 of 279

62

24 Years After Desert Storm: What Have We Learned About the Cause(s) of Gulf War Illness?

Etiology appears complex; important to evaluate health outcomes in veteran subgroups

Studies most consistently implicate anti-nerve gas pills (PB), and excess use of pesticides

low-level exposure to chemical weapons, oil fires, chemical combinations not ruled out

Chemical risk factors of greatest concern are toxic to the brain/nervous system. Many are in single class of toxicants (AChE inhibitors)

63

Question: Why did some veterans develop GWI, while others remained well?

Individual Differences in Vulnerability to Adverse Effects of Neurotoxicants? A number of circulating enzymes protect us from adverse effects of some neurotoxicants

(e.g. carbamates, OPs); long speculation that GWI risk may be associated with genetic variability in protective enzymes

Paraoxonase (PON1): Previous studies have suggested possible association of GWI with PON1 enzyme activity levels or genotype

Butyrylcholinesterase (BChE): Enzyme that binds acetylcholinesterase inhibitors, protects from adverse effects



Page 56 of 279

64

Paraoxonase (PON1) in Relation to GWI Studies Mixed; Overall Picture Difficult to Compare, Interpret

Haley et al 1999: Small study of Navy Seabees: PON1 activity (paraoxon) nonsign. higher in cases; type Q arylesterase activity sign. lower in cases. More cases than controls had PON1192 R allele.

Mackness et al 2000: 152 U.K. Gulf War veterans had lower PON1 activity (in paraoxon) than nondeployed comparison group; no genetic differences

Hotopf et al 2003: 210 U.K. Gulf War veterans had lower PON1 activity than nondeployed veterans; no association with illness, no genetic differences

Concato et al 2007: no PON1 activity differences in GWI cases vs. controls

Is GWI Associated with BChE Enzyme Activity or Genotype?



Page 57 of 279

Collaborators: Midwest Research Institute (now MRI Global) Antonio Sastre, PhD Mary Cook, PhD Mary Gerkovich, PhD Eppley Institute, University of Nebraska Medical School Oksana Lockridge, PhD Study funded by the U.S. Department of Defense

67

Butyrylcholinesterase (BChE): Overview

BChE: present at > 10X level of AChE in blood; physio functions not well understood.

Acts as scavenger, binds organophosphates, carbamates, other compounds

Early pharmacogenetics: Patients with abnormal response to succinylcholine (e.g. given for surgery) found to have inherited BChE deficiency. Produces prolonged paralysis/unable to breathe after dose that normally acts for few minutes

One BChE gene, multiple variations: Wild type “U” allele most common; then K, A, F

Reduced serum BChE activity routinely used as a biomarker for pesticide exposure

BChE currently being developed as a prophylactic measure to protect against effects of nerve agents (safer alternative to PB)



Page 58 of 279

68

Is GWI Associated with BChE?

Background Lowenstein-Lichtenstein (1995) [Soreq lab]: Case report of Israeli soldier—severe

symptoms with PB pills during the Gulf War; found to be BChE AA homozygote, poor BChE affinity for carbamates

Lockridge: 1999 DOD project: Nebraska GW veterans: most (73%) carriers of A, F alleles had reported they had Gulf War syndrome (vs < 30% overall)

69

Butyrylcholinesterase in Relation to Gulf War illness

Study Design

Assessed both BChE enzyme activity and genotype in 304 1991 Gulf War veterans

Population-based sample: 144 GWI cases (KS case def), 160 GW veteran controls

Determined if risk associated with “cholinergic exposures” (e.g. pesticides, PB, possible nerve agent exposure) differed with BChE genotype



Page 59 of 279

70

Study: Butyrylcholinesterase in Relation to Gulf War illness

BChE subgroup analyses: Compared variant subgroups: “common”

genotypes (UU and UK), “LCV--Less Common Variants” (K/K, U/AK, U/A, A/F, A/K,F) - “Normal” BChE variants: enzyme effective at neutralizing AChE inhibitors - “Less common” variants: enzyme acts more slowly, less effective at neutralizing some chemicals

Overall, BChE enzyme activity and genotype of GWI cases were similar to controls

71

Results: Butyrylcholinesterase Enzyme Activity Levels

n BChE Mean (SD)

Activity*

All veterans in study 304 1.10 (0.26)

BChE genotype

U/U 189 1.19 (0.24)

U/K 87 1.01 (0.21)

K/K 13 0.80 (0.15)

U/AK 10 0.76 (0.18)

U/A 3 1.03 (0.12)

A/F 1 0.92

AK/F 1 0.69

Common variants combined (U/U and U/K) 276 1.13 (0.24)

Less common variants combined (K/K, U/AK, U/A, A/F, AK/F)

28 0.81 (0.17)ꜛ

_______

Enzyme activity expressed in umoles benzoylcholine

hydrolyzed per minute per ml. serum



Page 60 of 279

72

Results: Butyrylcholinesterase Enzyme Activity Levels

Gulf War illness case status n

BChE Mean (SD) Activity

Gulf War illness cases 144 1.10 (0.24)

Gulf War veteran controls 160 1.10 (0.27)

_______

Enzyme activity expressed in umoles benzoylcholine

hydrolyzed per minute per ml. serum

73

Results: Distribution of Butyrylcholinesterase Genotype

Total Sample (n = 304)

GWI Cases (n = 144)

Controls (n = 160)

BChE Genotype

n

(%)

n

(%)

n

(%)

U/U 189 (62%)

89 (62%)

100 (62%)

U/K 87 (29%)

41 (28%)

46 (29%)

K/K 13 (4%)

7 (5%)

6 (4%)

U/AK 10 (3%)

5 (3%)

5 (3%)

U/A 3 (1%)

1 (<1%)

2 (1%)

A/F 1 (<1%)

0 (0%)

1 (<1%)

AK/F 1 (<1%)

1 (<1%)

0 (0%)

Less common variants combined (K/K, U/AK, U/A. A/F, AK/F)

28 (9%)

14 (10%)

14 (9%)



Page 61 of 279

74

Association of Gulf War Illness with Cholinergic Exposures, by BChE Genetic Subgroup

Experience/Exposure

All Gulf War Veterans

Took PB pills OR = 3.21*

Wore pesticide-treated uniforms OR = 3.72*

Used pesticides on skin OR = 2.89*

Living area sprayed with pesticides

OR = 1.33

Heard chemical alarms sounded OR = 1.31

*p< 0.001

Ref: Steele et al (2015) Environ Health 14:4

75


Experience/Exposure


BChE Common Variants (normal activity)

UU and U/K (n=276)

Took PB pills OR = 3.21* OR = 2.68*

Wore pesticide-treated uniforms OR = 3.72* OR = 3.63*

Used pesticides on skin OR = 2.89* OR = 3.14*


OR = 1.33 OR = 1.30

Heard chemical alarms sounded OR = 1.31 OR=1.26

*p< 0.001




Page 62 of 279

76


Experience/Exposure



UU and U/K (n=276)

BChE Less Common

Variants (slow-acting)

KK,UAK,UA,AF,AKF (n=28)

Took PB pills OR = 3.21* OR = 2.68* OR = 40.00*

Wore pesticide-treated uniforms

OR = 3.72* OR = 3.63* OR = 4.80

Used pesticides on skin OR = 2.89* OR = 3.14* OR = 1.33


OR = 1.33 OR = 1.30 OR = 1.64

Heard chemical alarms sounded OR = 1.31 OR=1.26 OR = 1.80

*p< 0.001


Preliminary Indication of Significant Gene-Exposure Interaction

Risk of Gulf War Illness in Relation to PB Use, by BChE Genetic Subgroup

1

6

11

16

21

26

31

36

"Normal" BChE Variants(UU, U/K BChE Genotypes)

Less Common BChE Variants(K/K,UAK,UA,AF,AKF)


Od

ds

Rat

io:

PB

Use

vs.

no

PB

Use

OR = 2.7*

OR = 40.0*



Page 63 of 279

78

Can Apparent BChE x PB Interaction be Explained by Study Factors?

Did BChE-LCV subgroup differ from other Gulf War veterans in other ways?

Demographically (sex, age, education) almost identical

Exposures: nearly identical (e.g. 58% vs. 57% reported PB use)

Was association with BChE an anomaly of KS GWI case definition?

Test by reassigning case/control status using CDC criteria Yields 187 CMI cases, 117 controls

79

Association of PB with Gulf War Illness in BChE Genetic Subgroups

Evaluated Using Two Case Definitions

Case Definition



UU and U/K (n=276)

BChE Less Common

Variants (slow-acting)

KK,UAK,UA,AF,AKF (n=28)

GWI—Kansas Case Definition OR = 3.21* OR = 2.68* OR = 40.00*

CMI—Fukuda/CDC Case Def OR = 1.99* OR = 1.73* OR = 11.37*

*p< 0.01




Page 64 of 279

80

Context

Study provides preliminary evidence that BChE-LCV subgroup (K homozygotes; A,F heterozygotes) who used PB were at substantially increased risk for GWI

Consistent with: 2 preliminary reports of BChE-associated risk in Gulf War veterans 2 rat studies indicate delayed neuro effects of low-dose PB only in strain with low BChE

enzyme activity

Caveats: Small sample (n=304) included only 28 with “at risk” genotypes

Hard to estimate the total number GWV affected by this interaction; based on

our study, this interaction would, at most, explain ~ 10 % of GWI cases

81

Conclusions

Study provides preliminary evidence of significant gene-exposure interaction; subgroup of Gulf War veterans with less active genetic forms of BChE who used PB were at significantly greater risk for GWI, compared to other veteran subgroups

Highly significant association, OR = 40 for BChE LCV subgroup Small (but representative) sample: association not explained by study factors Requires replication in larger sample

Analytic methods important: Factors related to possible genetic “vulnerability” only

apparent when exposures considered Additional BChE questions: - “Situational” reduced BChE activity in theater associated with GWI for other veterans? - Implications for animal models: species differences in detoxicating enzymes, including BChE



Page 65 of 279

82

Thank you

83

Approach used in BChE study may provide a foundation/model for further evaluation of gene-exposure interactions in GWI

As in other areas of Gulf War research, essential to evaluate appropriate subgroups

Genetic variants that confer reduced ability to neutralize certain chemicals would not be expected to yield poor health outcomes in the absence of those exposures

Prior PON1 studies did not evaluate association of GWI with genes in relation to wartime exposures; evaluated genotype, enzyme activity in:

GW deployed vs. nondeployed Veterans with GWI vs healthy controls (either GW vets or nondeployed)



Page 66 of 279

84

Paraoxonase (PON1)

Are differences in PON1 genotype associated with Gulf War Illness?

A Different Approach

85

Paraoxonase (PON1): Background

PON1: Hydrolyzes (inactivates) organophosphate (OP) compounds (AChE inhibitors), including insecticides and nerve agents

PON1 differences thought to confer differing sensitivity to specific OP compounds: PON1192 - Q variant most effective at hydrolyzing nerve agents PON1192 - R variant most effective at hydrolyzing some pesticides (e.g. chlorpyrifos, parathion)



Page 67 of 279

86

Exploratory GWI - PON1 Evaluation in a Case-Control Study

Population-based sample (n=72); “high risk” 1991 Gulf War veterans

40 GW veterans with GWI (GWI cases) 18 GW veteran controls 14 nondeployed/era veteran controls No case/control differences by sex, age, race, education

GW sample: GW veterans at highest risk for GWI All served in Army, enlisted ranks, forward deployed In units identified as potentially exposed to nerve agents

Highly exposed cohort; GWI sign. associated with degree of exposure to PB, pesticides

87

Association of GWI with GW Exposures, by PON1192 Genotype

ORs for GW Cases (n=40) vs. GW controls (n=18)

Experience/Exposure

All Gulf War

Veterans

Chemical alarms sounded OR = 1.90 (ns)

Used skin pesticides OR = 1.03 (ns)

Wore pesticide-treated

uniforms OR = 3.10 (ns)

Took PB 1 week or longer OR = 3.41*

_________ *statistically significant, p<0.05



Page 68 of 279

88

Association of GWI with GW Exposures, by PON1192 Genotype

ORs for GW Cases (n=40) vs. GW controls (n=18)

Experience/Exposure

All Gulf War

Veterans

PON1 QQ

Genotype

(n=31)

PON1 QR/RR

Genotypes

(n=27) sign.

intrxn?

Chemical alarms sounded OR = 1.90 (ns) OR = 0.75 (ns) OR = 7.56* ~

Used skin pesticides OR = 1.03 (ns) OR = 2.33 (ns) OR = 0.20 (ns) ~

Wore pesticide-treated

uniforms OR = 3.10 (ns) OR = 21.0*

[logit est] OR = 0.72 (ns) 0.02*

Took PB 1 week or longer OR = 3.41* OR = 11.20* OR = 0.74 (ns) 0.04*

_________ *statistically significant, p<0.05

89

Summary

Preliminary Evidence: Suggests Association of GWI with Exposures May Differ in Relation to PON1 Genotype

Recall PON1 R variant has better capacity for hydrolyzing some pesticides,

but provides poorer hydrolysis of sarin

Veterans with R allele were at sign. greater risk for GWI if they heard

chemical alarms (QQ homozygotes were not)

Use of pesticides and PB were sign. associated with GWI only in PON1 QQ

homozygotes (not in veterans with PON1 R allele)



Page 69 of 279

90

Preliminary Indication: Association of GWI with GW Exposures May Differ in Relation to PON1

Supports approach described for BChE study, i.e. that understanding impact of genetic vulnerability to neurotoxicants requires evaluation of GWI-exposure interaction in PON1 genetic subgroups

Caution: Small sample size; findings are exploratory

Identified subgroup associations are in directions that might be expected in relation to PON1 genotype



Page 70 of 279

92

EXTRA SLIDES

93

24 Years After Desert Storm: What Have We Learned About Gulf War Illness (GWI)?

Affects 25 – 30 % of the 700,000 U.S. personnel who served in 1991 Gulf War

GWI not due to stress or psych disorder; PTSD rates low in GW vets

No widespread “GWI”-type problem in OIF, OEF veterans

Higher GWI rates in ground troops, enlisted personnel, forward-deployed

Few veterans have recovered over time; many have been sick for ~ 24 yrs



Page 71 of 279

Table 3 Association of Gulf War illness with exposures with potential cholinergic effects, by butyrylcholinesterase genetic subgroup

Butyrylcholinesterase Genetic Subgroup

U/U Homozygotes

(n = 89 GWI cases, 100 controls)

U/K

Heterozygotes (n = 41 GWI cases, 46 controls)

Less Common BChE Variants (K/K, U/AK, U/A, A/F, AK/F)

(n = 14 GWI cases, 14 controls)

Experience/Exposure

%

Cases Exposed

% Controls Exposed

OR

*

(95% C.I.)

%

Cases Exposed

% Controls Exposed

OR

*

(95% C.I.)

%

Cases Exposed

% Controls Exposed

OR

*

(95% C.I.)

Took PB (pyridostigmine bromide) pills

67% 48%

2.25 (1.23–4.11)

74% 41%

3.98 (1.57-10.1)

92% 23%

40.0ꜛ

(3.58-447)

Wore uniforms treated with pesticides

22% 8%

3.07 (1.27-7.44)

37% 11%

4.92 (1.59-15.2)

29% 8%

4.80 (0.46-50.2)

Used pesticide cream or spray on skin

55% 31%

2.75 (1.50-5.02)

62% 28%

4.23 (1.71-10.5)

50% 43%

1.33 (0.31-5.91)


21% 18%

1.23 (0.59-2.57)

24% 17%

1.47 (0.51-4.29)

21% 14%

1.64 (0.23-11.7)

Heard chemical alarms sounded

57% 57%

1.02 (0.57-1.82)

62% 44%

2.00 (0.84-4.79)

64% 50%

1.80 (0.40-8.18)

BChE=butyrylcholinesterase, GWI=Gulf War illness, OR=prevalence odds ratio, C.I.=confidence interval *OR compares GWI cases with controls within each genetic subgroup

Table 4 Association of pyridostigmine bromide use with chronic illness in BChE subgroups: evaluation using two case definitions

Use of PB by Butyrylcholinesterase Genetic Subgroups

Use of PB by All Gulf War Veterans

(n = 304)

Common BChE Variants (U/U and U/K)

(n=276)

Less Common BChE Variants (K/K, U/AK, U/A, A/F, AK/F)

(n=28)

Case Definition

% Cases

Exposed

% Controls Exposed

OR (95% C.I.)

% Cases

Exposed

% Controls Exposed

OR (95% C.I.)

% Cases

Exposed

% Controls Exposed

OR (95% C.I.)

GWI (Kansas case definition)*

72% 44% 3.21

(1.97-5.24) 69% 46% 2.68

(1.62-4.44) 92% 23% 40.00

(3.58-447)

CMI (CDC case definition)ꜛ 63% 46%

1.99 (1.23-3.22)

62% 49%

1.73 (1.05-2.84)

78% 22%

11.37 (1.65-78.4)

BChE=butyrylcholinesterase, PB=pyridostigmine bromide, CDC=U.S. Centers for Disease Control and Prevention, OR=prevalence odds ratio, C.I =confidence interval *Gulf War illness, as defined in Steele [1]. ꜛChronic multisymptom illness, as defined in Fukuda et al [32].



Page 72 of 279

96

Gulf War Illness

Concurrent Symptoms in Multiple Domains

NEUROLOGICAL

SYMPTOMS

Memory Problems

Headaches

Dizziness

Mood Changes

RESPIRATORY

SYMPTOMS

Persistent Cough

Wheezing

GASTROINTESTINAL

SYMPTOMS

Diarrhea

Nausea

SKIN

PROBLEMS

Rashes

Other Problems

PAIN

SYMPTOMS

Joint Pain

Muscle Pain

FATIGUE

SYMPTOMS

Fatigue

Sleep Problems

Summary: Association of GWI with Deployment Experiences/Exposures Across GW Veteran Populations



Page 73 of 279

preliminary evidence of gene-exposure interaction in 1991 gulf … · preliminary evidence of...

Documents